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Abstract

Background

The Wnts are secreted proteins that play important roles in skeletal myogenesis, muscle
fiber type diversification, neuromuscular junction formation and muscle stem cell
function. How Wnt proteins orchestrate such diverse activities remains poorly understood.
Canonical Wnt signaling stabilizes β-catenin, which subsequently translocate to the
nucleus to activate the transcription of TCF/LEF family genes.

Methods

We employed TCF-reporter mice and performed analysis of embryos and of muscle groups.
We further isolated fetal myoblasts and performed cell and molecular analyses.

Results

We found that canonical Wnt signaling is strongly activated during fetal myogenesis
and weakly activated in adult muscles limited to the slow myofibers. Muscle-specific
transgenic expression of a stabilized β-catenin protein led to increased oxidative
myofibers and reduced muscle mass, suggesting that canonical Wnt signaling promotes
slow fiber types and inhibits myogenesis. By TCF-luciferase reporter assay, we identified
Wnt-1 and Wnt-3a as potent activators of canonical Wnt signaling in myogenic progenitors.
Consistent with in vivo data, constitutive overexpression of Wnt-1 or Wnt-3a inhibited the proliferation
of both C2C12 and primary myoblasts. Surprisingly, Wnt-1 and Wnt-3a overexpression
up-regulated BMP-4, and inhibition of BMP-4 by shRNA or recombinant Noggin protein
rescued the myogenic inhibitory effect of Wnt-1 and Wnt-3a. Importantly, Wnt-3a or
BMP-4 recombinant proteins promoted slow myosin heavy chain expression during myogenic
differentiation of fetal myoblasts.

Keywords:

Background

Skeletal muscles of the trunk and limb, except for some craniofacial and esophageal
muscles, are derived from somites during embryonic development [1-3]. Specification of somitic cells into myogenic lineages is regulated by positive and
negative signals from the surrounding tissues. Wnt signaling induced by Wnt-1, -3a,
-4, -6, -7a and 11 from dorsal neural tube or ectoderm is critical for the induction,
initiation and progression of myogenesis in the presomitic mesoderm and early somites
(Reviewed in [4,5]). Within the embryonic myogenic progenitors, Wnt also regulate the expression of
Pax3/7, MyoD and Myf5, key transcription factors involved in myogenesis [6-10]. Importantly, genetic knockout studies have clearly demonstrated the requirement
of several Wnt molecules and β-catenin in the normal development of skeletal muscles
[11,12]. These diverse functions of Wnt are mediated by both a canonical signaling pathway
that requires stabilization and nuclear translocation of β-catenin, and non-canonical
pathway that is independent of β-catenin [5]. Therefore, canonical and non-canonical Wnt signaling pathways play multiple essential
roles in embryonic myogenesis.

Myofibers in postnatal skeletal muscles retain an adaptive capacity to switch between
slow- and fast-twitch properties that largely depend on motoneuron activity [19]. Wnt signaling also regulates the establishment and maintenance of neuromuscular
junctions that connect motor neurons and myofibers. In Drosophila and mice, Wnt secreted
by presynaptic motoneurons interact with Agrin-MusK to induce assembly of postsynaptic
endplates (Reviewed in [24]). There is evidence that neuromuscular junctions are phenotypically and functionally
distinct in fast and slow muscles [25,26]. Whether Wnt signaling is differentially activated in slow and fast myofibers is
completely unknown.

In this study, we used transgenic Wnt-reporter mice and mice with constitutively activated
canonical Wnt signaling specifically within skeletal muscle to investigate the function
of canonical Wnt signaling in muscle. We found that Wnt signaling is highly activated
in prenatal muscle and rapidly declines in postnatal muscle, with some residual activation
limited to the slow myofibers. Interestingly, Wnt signaling is highly activated proximal
to motor endplates of slow myofibers. Wnt activation induced by stabilized β-catenin
inhibits muscle differentiation and promotes slow muscle determination. At the molecular
level, canonical Wnt signaling induces BMP-4, which promotes expression of slow MyHC.
These novel results demonstrate that interplay between Wnt and BMP signaling regulates
skeletal myogenesis and muscle fiber type.

Fetal myoblasts were prepared from the legs of Myf5-Cre/ROSA26-YFP embryos at E14.5.
The legs are minced and digested by collagenase, dispase and DNaseI, as previously
described for satellite cells isolation [30]. After collagenase procedure, collected cells were stained by alpha7-integrin antibody
and anti-mouse IgG1-Alexa648. Fetal myoblast were isolated by MoFlo (Dako, Glostrup,
Denmark). Fetal myoblast were isolated by magnetic-antibody cell sorting (MACS) (Miltenyi
Biotec, Bergish Gladbach, Germany). The isolated cells were incubated in culture dishes
at 37°C for one hour to remove adherent cells, and the nonadherent cells were collected.
The isolated fetal myoblasts cultured in DMEM/F10 medium with 20% FBS and bFGF.

Retrovirus and lentivirus infection

To prepare ecotropic retrovirus, Phoenix-eco packaging (kindly gifted from Dr. Gally
Nolan) cells were transfected with retrovirus vectors using GeneJuice (Novagen EMD
Chemicals, Madison, WI, USA). Viral supernatants were harvested 30 hours post transfection
and used to infect C2C12 cells in the presence of polybrene (Sigma, 8 mg/ml) for 12
hours. Infected C2C12 cells were then washed twice with phosphate-buffered saline
(PBS), maintained in growth media and were selected 24 hours post-infection with puromycin
(1.5 micro g/ml, Sigma). Lentivirus was packaged in 293T cells (ATCC CRL-11268) transfected
with the mouse BMP-4 or scramble shRNA pLKO.1-puro plasmids in addition to the pMD2.G
and psPAX2 plasmids. Lentivirus was concentrated by ultracentrifuge and resolved in
PBS(−) for virus infection. Infected C2C12 and primary myoblast cells were then washed
twice with PBS, and maintained in growth media.

Gene expression analysis

Total RNAs were prepared from C2C12 and myoblast cells by TRIzol (Life Technologies).
RNA samples were reverse transcribed using random hexamer and oligo dT mixed primers
with SuperScriptII enzyme (Life Techonologies) according to the manufacturer’s instructions.
Reverse transcription reactions were diluted (1:10) with 10 mM Tris, pH 8.0, yielding
master samples of reverse-transcribed products. Real-time PCR reactions are previously
described [27]. Real-time data were gathered using a system (MX4000; Agilent Technologies, Santa
Clara, CA, USA) over 40 cycles (30 s at 90°C, 60 s at 58°C and 30 s at 72°C) followed
by a denaturation curve from 54°C to 94°C in 30-s increments of 0.5°C to ensure amplification
specificity. Threshold cycle (Ct) values were calculated with the MX4000 software
(Agilent Technologies, Santa Clara, CA, USA) by using moving window aver-aging and
an adaptive baseline. Fold changes, other calculations and chart plotting were performed
in Microsoft Excel (Redmond, WA, USA). The sequence of PCR primers is listed in Additional
file 1: Table S1.

Animal care

Myf5-Cre [31] heterozygous mice were bred with ROSA26-YFP [32]. TCF-lacZ [33] and Ctnn1 exon3 floxed [34] mice were previously described. All mice are maintained inside a barrier facility,
and experiments were performed in accordance with the University of Ottawa regulations
for animal care and handling.

Immunofluorescence staining

C2C12 cells were incubated at 37°C for two hours with 10 μM BrdU, then washed with
PBS(−) and fixed with 2% PFA/PBS. The fixed cells were stained with DAPI for 15 minutes
at room temperature, washed with PBS(−), and refixed with 2% PFA for 5 minutes at
room temperature. The refixed cells were treated with 2N HCl for 20 minutes at room
temperature (RT), neutralized by washing with 0.1 M borate buffer pH 8.5. The cells
were permeablized with 0.2% Triton X-100 PBS(−), blocked with broking buffer, incubated
with anti-BrdU antibody for 2 to 12 hours. After staining with the primary antibody,
cells were washed with PBS(−), stained with anti-mouse IgG1-Alexa488, washed with
PBS(−) and mounted on slide glass with Dako mounting buffer. Myoblasts were fixed
in 4% PFA/PBS (−) for 5 minutes, blocked with 10% goat serum/PBS (−) for 10 minutes,
and stained with MyHC slow, MyHC fast, MyHC pan, anti-mouse IgG1-Alexa568, anti-mouseIgG2b-Alexa648
(Life Technologies) and DAPI (Sigma). Slides were mounted in SlowFade Light antifade
Kit Component A (Molecular Probes) and analyzed with a Bio-Rad confocal laser scanning
microscope (model MRC-1024) (Bio-Rad Laboratories, Hercules, CA, USA).

The isolated muscle tissues were placed directly into optimal cutting temperature
(OCT) compound and frozen in deep cold isopentane with ethanol and dry ice. The muscle
tissues were cut 16 μm by cryostat and dried in room temperature. The muscle sections
were incubated with NADH-TR staining solution (0.8 mg/ml NADH and 1 mg/ml NBT in 50
mM Tris–HCl (pH 7.6) at 37°C. The stained muscle sections were washed with deionized
water, unbound NBT was removed by acetone solution, then the sections were re-washed
with deionized water and mounted with a coverslip.

Luciferase assays

Myoblasts in 24-well plates were transfected with the plasmids indicated and 50 ng
pRL-PGK using Lipofectamine (Invitrogen). Transfected cells were harvested around
24 hours after transfection, and luciferase activities in the cell extracts were measured
according to the manufacturer's instructions (Promega, Fitcburg, WI, USA) in a luminometer
(Microplate luminometer LB96V (EG&G Berthold Technologies, Bad Wildbad, Germany).
Luciferase activities as indicated by arbitrary unit were normalized by sea urchin
luciferase activities in each sample. All experiments were repeated at least three
times, and the averages of more than three independent experiments with standard deviations
are shown as bars [35].

Results

Canonical Wnt signaling is activated during fetal myogenesis and reduced in adult
muscle

As the first step to investigate the function of Wnt signaling in myogenesis, we used
the TCF-lacZ transgenic reporter mouse to examine the activity of the canonical Wnt
signaling pathway in embryonic and adult muscles. The promoter of the LacZ transgene
(encoding β-galactosidase, β-gal) contains multimerized TCF binding sites [33], a key downstream effector of canonical Wnt signaling. We analyzed β-gal activity,
in embryonic and adult muscles by X-GAL staining (Figure 1). β-gal activity was detected in many muscles in E14.5 embryos (Figure 1A). Among those labeled, β-gal activity was particularly intense in both forelimb
and hind limb muscles (Figure 1B), ventral body wall muscles (Figure 1C), dorsal spinotrapezius (Figure 1D) and intercostal muscles. The number of muscles labeled with X-GAL was reduced in
the forelimb and hand limb in P0 fetus (Figure 1E, F). In the adult, β-gal activity was undetectable in EDL and TA, muscles that are
predominantly enriched with fast type myofibers, but was readily detected in part
of the diaphragm (Figure 1G) and soleus (Figure 1H), muscles that are known to be enriched with slow myofibers. These data indicate
that canonical Wnt signaling is strongly activated during fetal myogenesis and declined
in postnatal muscles with some residual activity in slow myofibers.

Interestingly, the β-gal activity in postnatal slow muscle was especially strong at
neuromuscular junctions (dark dotted staining patterns in the mid-belly of the muscles;
Figure 1G-H). To further examine this phenomenon, we isolated single myofiber from the soleus
muscles (containing about 50% slow and 50% fast myofibers) of Tcf-lacZ mice. The isolated
single myofibers were fixed and stained with α-bungarotoxin (BTX) and β-gal antibodies,
together with slow-MyHC or pan-MyHC antibodies (Figure 2A-D). Consistently, strong β-gal immunoreactivity was detected proximal to motor endplates
located within BTX stained neuromuscular junctions of type I fibers (Figure 2A, B), but not in type II myofibers (Figure 2C-D). Co-labeling whole mount muscle with slow and fast myosin heavy antibody confirmed
the specific activation of β-gal in the in slow myofibers (Figure 2E, F). Overall, 97% of the β-gal+ myofibers co-expressed the slow MyHC, where only 8% of the β-gal- myofibers co-expressed slow MyHC (Figure 2G). These data indicate that canonical Wnt signaling is highly activated at the neuromuscular
junction area specifically in slow myofibers of adult skeletal muscles.

Canonical Wnt signaling promotes formation of slow myofibers in vivo

To confirm the role of canonical Wnt signaling in muscle fiber type specification
in vivo, we took advantage of the Ctnnblox(ex3) transgenic mice in which the exon 3 of β-catenin (Ctnnb) gene flanked by LoxP sites [34]. The exon 3 encodes serine and threonine residues that are normally phosphorylated
GSK3β, leading to the proteasomal degradation of β-catenin. Upon Cre-mediated excision
of Ctnnb exon 3, β-cateninΔEx3 is prevented from degradation (stabilized) and, therefore, constitutively active.

We first used Myf5-Cre to induce β-cateninΔEx3 expression in myogenic progenitor cells. Myf5 is an early myogenic commitment marker
during embryonic myogenesis [36]. Myf5-Cre/Ctnnblox(ex3) mice die at E15.5 with extremely reduced muscle mass (data not shown), thus precluding
further analysis of myofiber types. We next used MCK-Cre to drive β-cateninΔEx3 expression only in differentiated muscle cells. As expected, we detected increased
activation of canonical Wnt signaling in vivo in transgenic mice carrying MCK-Cre, Ctnnblox(ex3) and TCF-LacZ alleles. β-gal activity was detectable in TA muscles of the MCK-Cre/Ctnnblox(ex3)/TCF-LacZ mice, but not the Ctnnblox(ex3) /Tcf-lacZ littermate controls (Figure 3A, B).

To identify the Wnt molecules that activate the canonical Wnt signaling pathway in
muscle, we co-transfected candidate Wnt plasmids with the TCF/LEF reporter Super TopFlash.
We found that Wnt-1 and Wnt-3a strongly activated (>400 times increase in luciferase
activity), Wnt-2 and Wnt-10b moderately activated (>20 times increase in luciferase
activity), and Wnt-2b and Wnt-4 weakly activated (>2 times increase in luciferase
activity) the canonical Wnt reporter (Additional file 2: Figure S1A). Other Wnts (Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-10a, Wnt-11)
had no effect on the activation of the Super TopFlash reporter (Additional file 2, Figure S1A). We, therefore, used Wnt-1 and Wnt-3a to activate the canonical Wnt
signaling in the following studies.

That canonical Wnt signaling induces osteogenic ALP expression suggests a potential
interaction between Wnt and BMP signaling pathways. We first examined BMP-4 gene expression
using quantitative RT-PCR (qPCR) given its role in osteogenesis. Indeed, BMP-4 mRNA expression was increased by more than five-fold in Wnt-1 and Wnt-3a overexpressing
C2C12 cells (Figure 4A). In comparison, Myf5 and MyoD expression was not affected by Wnt-1 and Wnt-3a (Figure 4A). To ensure that canonical Wnt signaling is involved in the induction of BMP-4,
we examined Axin2, a transcriptional target of β-catenin and canonical Wnt signaling
[38]. Wnt-1 and Wnt-3a overexpression led to over 40X increase in the expression Axin2 (Figure 4A).

We next asked if BMP-4 is necessary for pro-osteogenic effect of canonical Wnt signaling.
We used lentiviral shRNA mediated knockdown of BMP-4 in C2C12 cells. This approach resulted in nearly 80% reduction of BMP-4 transcripts (Figure 4B). Importantly, BMP-4 knockdown reduced Wnt-1 and Wnt-3a induced Akp4 (ALP gene) expression by more than 70% (Figure 4B). By contrast, Axin2 mRNA levels were not decreased following knockdown of BMP-4 (Figure 4B), suggesting that BMP-4 signaling does not affect canonical Wnt signaling.

We further investigated if the anti-myogenic effect of canonical Wnt signaling is
mediated by BMP-4 using the same shRNA knockdown approach. In the control groups (scrambled
shRNA), there were only a few MyHC positive myotubes in the C2C12 cells overexpressing
Wnt-1 (Figure 4C) and Wnt-3a (Figure 4C). Knockdown of BMP-4 remarkably increased the numbers of MyHC positive myotubes in Wnt-1 (Figure 4E) and Wnt-3a (Figure 4F) expressing C2C12 cells. Consistent with this observation, BMP-4 shRNA rescued MyHC protein expression in Wnt-1 and Wnt-3a overexpressing C2C12 cells
(Figure 4G). Moreover, BMP-4 shRNA increased the mRNA levels of Myogenin, MyHC-IIa, MyHC-IIb and MyHC-I (Figure 4H). To confirm the above observations, we used recombinant Noggin protein, an antagonist
of BMP, to block BMP activity. Noggin dose-dependently increased the number of MyHC
positive myotubes in Wnt-3a expressing C2C12 cells (Figure 4I-L). These data provide compelling evidence that canonical Wnt signaling inhibits
myogenic differentiation through inducing BMP-4 signaling.

Canonical Wnt signaling induces slow MyHC expression through BMP-4

To directly examine the function of canonical Wnt signaling in muscle fiber type specification,
we isolated embryonic myoblasts from E14.5 embryos by FACS. We employed positive selection
for Myf5 and α7-integrin expression of myogenic cells from Myf5-Cre/ROSA-YFP embryos (Additional file 6: Figure S5). The purity of isolated fetal myoblasts was confirmed by immunostaining
for Pax7 and desmin (Additional file 6: Figure S5). Embryonic myoblasts were cultured for one day before being induced to
differentiate with or without the addition of Wnt-3a protein (50 ng/ml). In the absence
of Wnt-3a, embryonic myoblasts differentiated into myotubes that expressed fast MyHC
(Figure 5A), but not slow MyHC (Figure 5C) in agreement with previous studies [39]. By contrast, Wnt-3a treated fetal myoblasts differentiated into myotubes that expressed
both fast (Figure 5B) and slow MyHC (Figure 5D).

Additional file 6: Figure S5. Isolation of fetal myoblasts from E14.5 embryos of Myf5-Cre/ROSA26-YFP mice by fluorescence
activated cell sorting (FACS). (A-C) YFP expression in whole embryo (A), forelimb (B) and hindlimb (C) of E14.5-15.5 Myf5-Cre/ROSA26-YFP embryo. (D) Strategy for isolating fetal myoblasts by FACS. Whole limbs of Myf5-Cre/ROSA26-YFP
embryos were minced and digested by collagenase and dispase. Single cells were stained
with alpha7-integrin antibody and selected by YFP and alpha7-integrin expression.
The sorted YFP and alpha7-integrin double positive cells were stained with antibody
for Pax7 and desmin, makers of fetal myoblasts (nuclei were counterstained by DAPI
in blue).

To confirm this result, we analyzed MyHC-I mRNA expression by qPCR. The MyHC-I mRNA level was up-regulated 5.5-fold by Wnt-3a compared to control vehicle treatment
(Figure 5E). Wnt3a also robustly induced the expression of slow MyHC-I at the protein level
(Figure 5F). These results indicate that canonical Wnt signaling induces slow MyHC expression
in fetal myoblasts.

As canonical Wnt signaling induced BMP-4, we further examined the role of BMP-4 in
muscle fiber type specification. Consistent with our previous results in C2C12 cells
(Figure 4A), Wnt-3a treatment of embryonic myoblasts induced a seven-fold increase in BMP-4 mRNA expression (Figure 5E). Next, we added recombinant BMP-4 to fetal myoblast cultures during differentiation.
In the control treated with vehicle medium, newly formed MyHC+ myotubes seldom expressed slow MyHC after three days of differentiation (Figure 5G). In the presence of 2.5 to 5 ng/ml BMP-4, slow-MyHC+ myotubes were abundantly visible (Figure 5I-J). The level of slow MyHC immunofluorescence induced by 5 ng/ml BMP-4 is similar
to that induced by 50 ng/ml Wnt-3a (Figure 5H), suggesting BMP-4 more potently induces slow MyHC expression. Western blotting
showed that the slow MyHC protein expression level was indeed increased in the presence
of BMP-4 (Figure 5F). In addition, qPCR analysis indicated that BMP-4 not only induced the slow MyHC-I gene expression, but also robustly suppressed the fast MyHC-IIb gene expression (Figure 5K). Collectively, these results indicate that canonical Wnt signaling acts through
BMP-4 to induce slow MyHC expression during embryonic myoblast differentiation.

Discussion

In this study, we use genetic, cell culture and molecular biology approaches to dissect
the function of canonical Wnt signaling in myogenic differentiation and skeletal myofiber
types. We show that the canonical Wnt signaling is most active during perinatal myogenesis
and only activated in slow myofibers with high activity at the neuromuscular junction
area in mature muscles. Constitutive activation of β-catenin, the canonical Wnt signaling
effector, leads to impaired myogenesis and an increased proportion of oxidative myofibers
in the postnatal muscles. Importantly, Wnt-1 and Wnt-3a mediated downstream signaling
activates BMP-4, which inhibits the overall proliferation of myoblasts and promotes
myogenic differentiation towards slow muscle phenotype. These results establish a
novel interaction between Wnt and BMP signaling that regulates muscle fiber type specification
and maintenance.

The TCF-LacZ reporter mouse has been widely used in reporting activation of canonical
Wnt signaling in various tissues/cells [11,40]. Strong LacZ expression in specific muscles during embryonic and fetal myogenesis
indicates activation of canonical Wnt signaling. Interesting, several muscles (spinotrapezius,
body wall muscle and diaphragm) with high β-gal activity are known to be enriched
with slow myofibers [19]. These results suggest a role of Wnt signaling in slow muscle generation and maintenance.
Our in vivo results are consistent with previous studies in chick and fish embryos, in which
canonical Wnt signaling was shown to promote slow muscle fate [20-22].

Our analysis of canonical Wnt signaling in adult mature muscles reveal several interesting
points. First, β-gal activity is only detectable in muscles known to contain slow
myofibers. This result confirms our observation in the developing embryonic muscles.
Second, in contrast to embryonic muscle, where β-gal activity is evenly distributed
within myofibers, the highest β-gal activity was within the slow myofibers was proximal
to the motor endplate (Figure 1G-H). This observation suggests that whereas in embryonic muscle the Wnt molecules
are released from surrounding tissues [5], Wnt signaling in adult slow myofibers is most likely initiated by Wnt molecules
from motor neurons that innervate these myofibers. In support of the notion that the
motor neuron supplies Wnt molecules, we found that β-gal immunoreactivity was no longer
detectable in slow myofibers after three days of suspended culture in vitro in the absence of neural innervation (data not shown).

Previous studies demonstrate that Wnt molecules released by motor neurons play key
roles in the development of neuromuscular junctions. Specifically, interaction of
Wnt and LRP is necessary for clustering of postsynaptic acetylcholine receptors (AchR)
[24]. Our new results demonstrate that Wnt signaling is further required for the maintenance
of neuromuscular junction in slow myofibers. Future study is needed to examine the
functional significance of Wnt signaling in slow versus fast muscle fibers and identify
the Wnt molecules released by slow and fast motor neurons.

Using Cre-inducible transgenic mice that express stabilizes β-catenin, we investigated
the role of canonical Wnt signaling in embryonic myogenesis and postnatal muscle maintenance.
When Myf5-Cre is used as the driver mouse, which is expressed in embryonic myogenic
progenitors, we detected abnormal muscle development and perinatal lethality. This
observation is consistent to recent studies using Pax7-Cre or Myogenin-Cre to stabilize
β-catenin, which also results in lethality at P0 [23,41]. In these studies, constitutive activation of β-catenin in the myogenic progenitors
and committed myocytes resulted in a shift of fetal myofibers to slow muscle phenotype,
and reduced myofiber size. However, the perinatal lethality of the Myf5-Cre, Myogenin-Cre
and Pax7-Cre drive β-catenin activation precludes analysis of postnatal muscles. Using
the Mck-Cre/ctnnbLox(ex3) mice, we found that adult muscles indeed have higher canonical Wnt activity based
on the TCF-LacZ reporter assay. This verifies the utility of our mouse model.

Importantly, we found the adult fast (TA) muscles exhibited features of slow muscle
phenotype as increased oxidative capacity. We also examined myosin heavy chain expression
by immunohistochemistry but did not find any overt changes in myofiber types (data
not shown). This result suggests that although Wnt signaling affects metabolic properties
in the adult muscles, it is not sufficient to switch myosin heavy chain expression
in the adult. This is expected since other factors, such as hormones and neural activity,
can also influence myosin expression [19]. Together, our Mck-Cre/ctnnbLox(ex3) model bypasses the premature lethality and provides novel insights of canonical Wnt
signaling in regulating the oxidative capacity of adult muscles.

The observation that C2C12 cells overexpressing Wnt-1 and Wnt-3a exhibited reduced
proliferation and myogenic differentiation is quite intriguing. It could suggest that
canonical Wnt/β-catenin inhibits the proliferation and differentiation of myogenic
cell lineages. This possibility would explain the reduced muscle mass phenotypes of
the Myf5-Cre, Myogenin-Cre and Pax7-Cre induced stabilized β-catenin mice [23,41]. Alternatively, the result may also suggest that expression of Wnt-1 and Wnt-3a in
the cell, independent of Frizzled receptor activation, is detrimental to cell growth
and differentiation.

Interestingly, embryonic and fetal myoblasts seem to have different responses to canonical
Wnt signaling [23], suggesting that the role of Wnt signaling, even in the same cell lineage, is also
context dependent. Consistent with this notion, we show that the growth and differentiation
of fetal primary myoblasts are not inhibited by recombinant Wnt-3a protein in culture.
The osteogenic fate choice of Wnt-1 and Wnt-3a overexpressing C2C12 cells is in line
with a recent report demonstrating fibroblastic lineage differentiation of satellite
cells in response to high level of systemic Wnt molecules [18]. Thus, the observed effect of Wnt-1 and Wnt-3 in myogenic cell proliferation and
differentiation is largely consistent with information in the literature.

We discovered a novel interaction between Wnt and BMP signaling in myoblasts. Bone
morphogenetic proteins (BMPs) are multi-functional proteins belonging to the transforming
growth factor beta (TGFβ) superfamily. In zebrafish and frogs, BMP signaling inhibits
the differentiation of muscle precursors in the dermomyotome and controls the number
of myogenic cells [42,43]. During late myogenesis of mice, BMP signaling regulates the number of fetal myoblasts
and satellite cells [44]. This action is through preventing the premature activation of MyoD while maintaining
Pax3 expression. Therefore, BMPs may function to establish a sufficient number of
myogenic progenitors before terminal differentiation.

Our cell culture results indicate that Wnt signaling induces BMP4, and BMP4 inhibition
rescues the inhibitory effect of Wnt-1 and Wnt-3a on myogenesis. This result is in
line with the above results in vivo. We further identify an unexpected role for BMP-4 in promoting slow muscle fate during
fetal myogenesis. It is important to mention that low concentrations (1 to 5 ng/ml)
of BMP-4 protein were used in our study. Non-physiological, high concentrations of
BMP will probably generate completely different effects [45]. Future studies should illustrate how BMP signaling regulates myosin gene expression.

Interestingly, in Drosophila larval neuromuscular junctions, retrograde BMP signaling controls synaptic growth
[46]. The muscle-derived BMP modulates cytoskeletal dynamics and structural changes at
presynaptic terminals. This forms a feedback system in which canonical Wnt molecules
secreted from motor neurons not only induce formation of neuromuscular junctions,
but also activate BMP-4 expression in the muscle. The muscle derived BMP-4 subsequently
promotes development of presynaptic motor neuron terminals. Indeed, β-catenin stabilization
in skeletal muscles (not limited to the neuromuscular junction area) results in increased
motor axon number and excessive intramuscular nerve defasciculation and branching
[41]. Taken together, our experiments have identified a novel interaction between canonical
Wnt and BMP signaling that plays a role in myofiber type specification.

Conclusion

Our study demonstrates that canonical Wnt-signaling controls the development of skeletal
muscles via BMP-4 expression. High concentrations of BMP-4 have been previously established
to inhibit myogensis and induce osteogenesis. We found that isolated fetal myoblasts
do not normally form slow myofibers during myogenic differentiation in vitro. Strikingly, canonical Wnt-signaling induced low level BMP-4 expression that act
to induce slow myofibergenesis. Therefore, we conclude that canonical Wnt and BMP
signaling plays a hitherto unappreciated role in myofiber type specification during
fetal myogenesis.

Competing interests

The authors declare no competing interests.

Authors’ contributions

KK and MAR designed the research and wrote paper. SK performed the histology and tissue
staining, and helped with paper writing. TMM provided the Ctnn1 exon3 floxed mice.
All authors read and approved the final manuscript.

Acknowledgments

We thank Dr. Daniel Dufort for TCF-lacZ mice, Dr. Randall Moon and Dr. Valerie Wallace
for DNA constructs. We thank members of the Rudnicki laboratory for their technical
assistance and helpful discussions, and Dr. Makoto Sato for critical reading of the
manuscript. KK was supported by a Postdoctoral Fellowship from Training Program in
Regenerative Medicine in Canada.